Expression of a granule membrane marker on the surface of neutrophils permeabilized with digitonin. Correlations with...

The authors have previously shown that human neutrophils can be permeabilized with the cholesterol-complexing agent digitonin and that these cells can be induced to secrete granule contents by increasing free Ca2+ concentrations. In the studies reported here, the authors wished to determine whether secretion of granule constituents correlated with the appearance of an immunologic marker for granule membranes on the surface of the permeabilized neutrophils. For this purpose, we used flow cytometry and two fluorometrically identifiable markers, Mo1 (a granule membrane marker) and beta 2-microglobulin (beta 2m) (a plasma membrane marker). It was found that the ratio of Mo1/beta 2m increased for permeabilized neutrophils which were exposed to micromolar concentrations of free Ca2+. This increase in the detectable surface concentration of Mo1 was accompanied by the release of lysozyme, vitamin B12 binding protein, and beta-glucuronidase into the medium. Statistical analysis showed a very strong correlation between granule secretion and the Mo1/beta 2m ratio. These data thus suggest that granule membrane components were being introduced into the plasma membrane during Ca2+-induced granule discharge; this in turn suggests that secretion by permeabilized neutrophils represents true degranulation.